Figure 1.
High-fat diet induce obesity, hepatic steatosis, and increased hepatic cytokine expression.
Wild-type C57BL/6 mice were fed either normal diet or high-fat diet for 12 or 16 weeks.(A) Animal weight, (B) Liver histology (200x magnification), and (C) Hepatic cytokine expressions, determined by quantitative real time PCR. Values are mean ±SEM, * P<0.05,**P<0.01 versus NC; NC: normal diet control, HF:high fat diet; n=5 animals per group.
Figure 2.
High-fat diet increase Kupffer cells and decrease NKT cells.
Wild-type C57BL/6 mice were fed either normal diet or high-fat diet for 12 or 16 weeks. Kupffer cells were determined by immunohistochemistry staining or isolated through collagen perfusion. Hepatic mononuclear cells (HMNC) were isolated and NKT cells were identified with CD1d tetramer and CD3 double positive cells by flowcytometry. (A) F4/80 positive cells (Kupffer cells) distribution detected by immunohistochemistry (200x magnification), (B) Semi-quantitation of F4/80 positive cells, sections from mice fed either NC or HF were stained with F4/80.Ten fields per section from each mouse were analysed. (C) The numbers of isolated Kupffer cells form NC or HF feeding. (D) Representative dot plots of hepatic NKT cells (CD1d tetramer+CD3+) , (E) Mean (±SEM) results of hepatic NKT cell percentage. Values are mean ±SEM, * P<0.05,**P<0.01 versus NC, n=4 animals per group.
Figure 3.
Cytokine gene expression in Kupffer cells after lipid treatment.
(A) Cytokine gene expression on Kupffer cells after high-fat diet feeding. Wild-type C57BL/6 were fed either normal diet or high-fat diet for 16 weeks. Kupffer cells were isolated and cytokine gene expression were determined by quantitative real-time PCR. (n=4/group). (B) Cytokine expression on Kupffer cells after certain fatty acids treatment. Kupffer cells isolated from normal diet mice were treated with certain fatty acids (PA ,OA) for 24 h. (n=3/group). (PA,OA, 0.5mmol/L), Values are means±SEM, *P<0.05,**P<0.01 versus normal control.
Figure 4.
TLR4 expression in Kupffer cells after lipid treatment.
(A) High-fat diet induced increased expression of TLR4 in Kupffer cells. (B) Fatty acids treatment induced increased expression of TLR4 in Kupffer cells. Values are means±SEM, *P<0.05 versus normal control, (n=4/group).
Figure 5.
Kupffer cell present exogenous but not endogenous antigen to NKT cell.
Kupffer cells and hepatocytes were isolated from wild-type mice. (A) Representative histogram of Kupffer cell CD1d expression. Green line indicates staining with isotype control. (B) Endogenous antigen presentation of hepatocytes and Kupffer cells. Kupffer cells and hepatocytes were isolated and co-cultured with a NKT cell line. IL-2 released from NKT cells indicated activation of NKT cells by hepatocytes or Kupffer cells. IL-2 levels in culture supernatant were determined by ELISA. (**P<0.01 versus NKT alone). (C) Exogenous antigen presentation of Kupffer cells. Kupffer cells were isolated and loaded with αGalCer (100ng/ml), then co-cultured with NKT cell line for 24h. IL-2 levels in culture supernatant was determined. (**P<0.01 versus unload Kupffer cell group). (D) Comparion of ability of exogenous lipid antigen presentation of hepatocytes and Kupffer cells. (*P<0.05 versus hepatocyte group) . Values are means±SEM, n=3/group.
Figure 6.
Fatty acids treatment increase Kupffer cells ability on antigen presentation and activation of NKT cells.
Kupffer cells were isolated from wild-type C57BL/6 mice and treated with fatty acids (PA, 0.5mmol/L) and loaded with αGalCer (100ng/ml) overnight, and co-cultured with HMNC (served as NKT cells source) for 24h. Cells and culture supernatant were collected for detection of cytokine mRNA expression and protein levels. Increased expression of IFN-γand IL-4 indicated activation of NKT cells by αGalCer-loaded Kupffer cells. (A) IFN-γand IL-4 mRNA expression in co-culture system determined by real time PCR. (B) IFN-γand IL-4 levels in culture supernatant determined by ELISA. Values are means±SEM , *P<0.05, **P<0.01 versus Blank, #P<0.05 αGalCer group versus αGalCer+PA group, n=3 experiment.
Figure 7.
LPS had synergistic effect on Kupffer cells ability of activation of NKT cells.
Kupffer cells were isolated from wild-type C57BL/6 mice and treated with fatty acids (PA 0.5mmol/L) and/or LPS (100ng/ml) in the presence of αGalCer (100ng/ml) overnight, then co-cultured with HMNC for 24h. (A) IFN-γand IL-4 mRNA expression in co-culture system. (B) IFN-γand IL-4 levels in culture supernatant determined by ELISA. Values are means±SEM , *P<0.05 versus control, #P<0.05 versus respective control , n=3 experiment.
Figure 8.
Pro-inflammatory activated Kupffer cells by lipids lead to NKT cells activation- induced cell death.
Kupffer cells and HMNCs co-culture system was established as above. After co-culture for 20h, cells were collected and stained with fluorescent antibodies and determined by flow cytometry. Necrosis and apoptosis of NKT cells (gate from CD3+NK1.1+ cells) was defined as Annexin V+7AAD+ cells and Annexin V+7AAD- cells respectively. (A) Representative dot plots of NKT cells (NK1.1+CD3+) necrosis and apoptosis, (B) Mean (±SEM) results of percentage of NKT cell death (apoptosis plus necrosis). *P<0.05 versus Blank, #P<0.05 αGalCer group versus αGalCer+PA group, n=3 experiment.